Expression Analysis and Biological Characterization of Babesia Sp. BQ1 (Lintan) (Babesia Motasi-Like) Rhoptry-Associated Protein

Niu et al. Parasites & Vectors (2016) 9:313 DOI 10.1186/s13071-016-1573-7

RESEARCH Open Access Expression analysis and biological characterization of Babesia sp. BQ1 (Lintan) (Babesia motasi-like) rhoptry-associated protein 1 and its potential use in serodiagnosis via ELISA Qingli Niu1, Zhijie Liu1, Jifei Yang1, Peifa Yu1, Yuping Pan1, Bintao Zhai1, Jianxun Luo1, Emmanuelle Moreau3, Guiquan Guan1* and Hong Yin1,2*

Abstract Background: In China, ovine babesiosis is one of the most important tick-borne haemoparasitic diseases of small ruminants. It has a significant economic impact, and several Babesia motasi-like isolates have been recently shown to be responsible for ovine babesiosis in this country. Methods: Full-length and C-terminal-truncated forms of the rap-1a61-1 gene of Babesia sp. BQ1 (Lintan) were cloned into the pET-30a plasmid and subsequently expressed as His-fusion proteins. The resulting recombinant RAP-1a proteins (rRAP-1a61-1 and rRAP-1a61-1/CT) were purified and evaluated as diagnostic antigens using Western blot analysis and ELISA. The native Babesia sp. BQ1 (Lintan) RAP-1 protein was recognized using Western blots and IFAT by antibodies that were raised in rabbits against rRAP-1a61-1/CT. The specificity, sensitivity and positive threshold values for rRAP-1a61-1/CT in ELISA were evaluated. Results: Cross-reactivity was observed between rRAP-1a61-1/CT and positive sera for Babesia sp. BQ1 (Lintan), Babesia sp. BQ1 (Ningxian) and Babesia sp. Tianzhu isolates obtained from infected sheep. At one week post- inoculation, a significant increase was observed in the amount of antibodies produced against RAP-1a, and high levels of antibodies against RAP-1a were observed for 3 months (at 84 days p.i.). A total of 3198 serum samples were collected from small ruminants in 54 different regions in 23 provinces of China. These samples were tested using ELISA based on the rRAP-1a61-1/CT protein. The results indicated that the average positive rate was 36.02 %. Conclusions: The present study suggests that rRAP-1a61-1/CT might be a potential diagnostic antigen for detecting several isolates of B. motasi-like parasites infection. Keywords: Babesia sp. BQ1 (Lintan), Rhoptry-associated protein, Biological characterization, Diagnosis

* Correspondence: [email protected]; [email protected] 1State Key Laboratory of Veterinary Etiological Biology, Key Laboratory of Veterinary Parasitology of Gansu Province, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Science, Xujiaping 1, Lanzhou, Gansu 730046, P. R. China Full list of author information is available at the end of the article

© 2016 Niu et al. Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Niu et al. Parasites & Vectors (2016) 9:313 Page 2 of 14

Background motasi-like, B. orientalis and B. gibsoni) [12, 18–23]. RAP- Ovine babesiosis is one of the most important tick- 1 was detected at all asexual growth stages [24] and was borne diseases of small ruminants. This disease has been expressed during the sporozoite stage by a RAP-1 specific described in many countries throughout Asia, Europe and antisera that was obtained from rabbits immunized to Africa [1]. A phylogenetic tree based on the sequence of neutralize the binding of sporozoites of B. bovis to eryth- the 18S rRNA gene showed that Babesia species belong- rocytes [25]. Moreover, the role of RAP-1 in the erythro- ing to the following four groups are responsible for cyte invasion process has been studied in vitro during babesiosis in small ruminants: B. ovis, B. motasi which are parasite adhesion to erythrocytes, and experiments have a European and a Chinese clade (B. motasi-like), and the been performed to study the inhibitory effect of RAP-1- Babesia sp. Xinjiang and B. crassa [2–7]. In the B. motasi specific antibodies on growth and invasion [24–26]. Chinese clade (B. motasi-like), six Babesia isolates (Babe- Immunization with native and recombinant RAP-1 pro- sia sp. BQ1 (Lintan), Babesia sp. BQ1 (Ningxian), Babesia tein from B. bovis and B. bigemina also induced immunity sp. Tianzhu, Babesia sp. Hebei, Babesia sp. Madang and and protected animals upon challenge [27–29]. These Babesia sp. Liaoning) have been recently reported in the studies strongly support the notion that RAP-1 plays a Gansu, Hebei and Liaoning provinces of China. These functional role in the biology and development of Babesia isolates can be classified into one or more sub-groups ac- parasites and represents a potential candidate for the de- cording to their phylogenetic relationships, which have velopment of recombinant vaccines against babesiosis been reconstructed based on several (18S rRNA, ITS, 28S, [30–33]. rap-1) phylogenetic markers [7–12]. Rap-1 was characterized and found to be present as Currently, the strategy for controlling babesiosis is based multiple gene copies that are arranged in a tandem, head on chemotherapy against either the vector or the parasite. to tail manner in all Babesia species. In sheep Babesia Several compounds are used, but mainly ivermectin is species, the rap-1 locus of the B. motasi-like gene is used against ticks and imidocarb against Babesia. Several similar to the locus of B. bigemina: in both species, three new anti-Babesia drugs (Triclosan, Nerolidol, Artesunate different isoforms (rap-1a, rap-1b and rap-1c), 5 identi- and Atovaquone) are also used to control and treat cal rap-1b copies and a single rap-1c located at end of babesiosis [13]. However, this strategy has many draw- locus have been described [12, 34]. Two rap-1a classes backs, such as drug-resistance, the presence of drug named rap-1a61 and rap-1a67 have been described, and metabolites in the milk and meat, and high toxicity to the each class is represented by two close variants (rap- animals themselves. Optimal treatment methods, includ- 1a61-1 and rap-1a61-2; rap-1a67-1 and rap-1a67-2)in ing those with biological activity against ticks and vaccines Babesia sp. BQ1 (Lintan) [34]. Polymorphisms were lim- that target either the ticks or the parasites, have been ited to three nucleotide positions in the rap-1a61 copies extensively studied. But only live attenuated vaccines are (rap-1a61-1, -2, -3) and were found in the rap-1 locus of currently used in some countries, and no producing or Babesia sp. BQ1 (Lintan) (rap-1a61-1 and -2), Babesia testing recombinant or subunit vaccines have been avail- sp. BQ1 (Ningxian) and Babesia sp. Tianzhu (rap-1a61- able to prevent babesiosis so far [13–16]. 2 and -3) [12, 34]. According to a phylogenetic tree that Clinical symptoms of babesiosis usually include fever, was constructed based on the rap-1 gene sequences, the anemia, and hemoglobinuria and can lead to death in B. motasi-like group can be separated into two sub- severe cases, which become apparent when merozoites groups: one that consists Babesia sp. BQ1 (Lintan and from the Babesia invade and replicate within host eryth- Ningxian) and Babesia sp. Tianzhu and another that rocytes, resulting in detectable parasitemia [17]. During contains Babesia sp. Hebei [12]. the process of erythrocytic invasion, some of the pro- The rap-1a gene has been found in all Babesia species teins, including apical membrane antigen-1 (AMA-1), and shows highly conserved protein features (30–45 % rhoptry-associated-protein-1 (RAP-1), and spherical identity among species), suggesting that this gene plays body proteins (SBP-1, 2, 3), are secreted by the apical an important role in the RBC invasion process [35]. organelles (rhoptries, micronemes and dense granules). Moreover, RAP-1a, in Babesia has been widely used as a Although the functions and implications of these pro- vaccine candidate and diagnostic antigen [21, 29, 36, 37]. teins for vaccines designed against Babesia infections The expression, biological characterization and immuno- have been described mainly in B. bigemina and B. bovis, genicity of RAP-1a in sheep Babesia species have not these data may form a basis for developing a recombin- been extensively studied. In the present study, we cloned ant vaccine against other Babesia infections [17]. and expressed RAP-1a and then detected the presence RAP-1 is an immunogenic protein that is approximately of antibodies directed against native and recombinant 40–60 kDa in size. It is localized in rhoptries and has been forms of RAP-1a during the course of infection. To characterized in all Babesia species examined so far (B. evaluate the potential of using recombinant RAP-1a as a bovis, B. bigemina, B. divergens, B. canis, B. ovis, B. diagnostic antigen, we compared the diagnostic potential Niu et al. Parasites & Vectors (2016) 9:313 Page 3 of 14

of full-length and truncated C-terminal recombinant A total of 3198 serum samples were randomly col- Babesia sp. BQ1 (Lintan) RAP-1a proteins using via lected from clinically healthy sheep from 54 different Western blot analysis. We discuss our results in combin- locations in 23 Chinese provinces between 2010 and ation with the outcomes of previous studies. 2015 (Fig. 1). All blood samples were collected in non- anticoagulation tubes and transported to the laboratory Methods while on ice. The serum was then separated and stored Parasites at -20 °C until further use. The original isolates of the B. motasi-like group, including Babesia sp. BQ1 (Lintan), Babesia sp. BQ1 (Ningxian), Ethical approval Babesia sp. Tianzhu and Babesia sp. Hebei, were obtained This study was approved by the Animal Ethics Commit- from infected sheep and then cryopreserved in liquid tee of the Lanzhou Veterinary Research Institute at the nitrogen at the Vector and Vector-borne Disease (VVBD) Chinese Academy of Agricultural Sciences. All animals Laboratory of the Lanzhou Veterinary Research Institute used in this study were handled in accordance with the (LVRI) (CAAS Lanzhou, China) [3, 4, 6]. Animal Ethics Procedures and Guidelines of the People’s Republic of China. Sera Positive sera were obtained from five different breeds of Cloning of the full-length and the C-terminal truncated sheep (two “Tan mutton” Chinese sheep, including No. RAP-1a61-1 genes 3216 and 2007, and three “Vendean” French sheep, in- Genomic DNA was extracted from infected blood sam- cluding No. 3309, 3533 and 3446) that were infected ples obtained from sheep infected with Babesia sp. BQ1 with Babesia sp. BQ1 (Lintan). Immune-positive sera (Lintan) using a QIAamp DNA Blood Kit (QIAGEN, were obtained from sheep Nos. 08026, 08040 and 08020, Maryland, USA) according to the manufacturer’s in- which were infected by Babesia sp. BQ1 (Ningxian), structions. These DNA samples were used as the tem- Babesia sp. Tianzhu and Babesia sp. Hebei, respectively, plates for PCR. The oligonucleotide primers that were and immune-positive sera were obtained from sheep No. used to amplify the full-length and C-terminal-truncated 3201, which was infected with Babesia sp. Xinjiang, as rap-1a61-1 genes were designed based on the DNA se- previously described [38]. These serum samples were quence of Babesia sp. BQ1 (Lintan) RAP-1a61-1 (Gen- used to evaluate cross-infection and antibody kinetics. Bank accession number KC953701) and modified to Positive sera from sheep that were experimentally in- include either Xba I and BamH IorNde I and Hind III fected with Theileria luwenshuni, T. uilenbergi and Ana- (New England BioLabs) enzyme restriction sites (under- plasma ovis or bovine B. bovis and B. bigemina were lined below) at their 5' end. The following primers were used as the controls to evaluate cross-reactivity. used: RAP-1a61-full-F: 5'-CGC TCT AGA -GT ACG CCA Fifty serum samples were collected from five sheep (3– TTA CCA CCG TTC-3', RAP-1a61-full-R: 5'-GCC GGA TCC 12 wpi) that were experimentally infected with Babesia sp. GAG CCA CGA ATC ATC GGA TTC-3', RAP-1a61/CT- BQ1 (Lintan). In total, 141 sera and anticoagulated blood F: 5'-CGC CAT ATG ACC ACG GTG ATG CCC GAG-3', samples were collected from sheep in the Xinjiang, Henan and RAP-1a61/CT-R: 5'-GCC AAG CTT GAG CCA CGA and Gansu provinces of China. Genomic DNA was puri- ATC ATC GGA-3'. The PCR products were cloned into fied from blood samples and tested using specific PCR the pUC57 vector according to the manufacturer’s instruc- primers, and 141 genomic DNA samples were found to be tions (Genscript). The inserts were sequenced using vector specifically positive for Babesia sp. BQ1 (Lintan) in the primers. Representative plasmids were selected and sequencing analysis (data not shown). The sera sam- digested using either Xba IandBamH IorNdeI and Hin- ples (n = 141) that corresponded to these genomic dIII restriction enzymes and subsequently cloned into DNA samples were selected as the positive serum digested pET-30a expression vectors (Genscript) according samples. A total of 191 sera were used as positive to the manufacturer’s instructions. This resulted in the samples to evaluate the sensitivity of the ELISA, and generation of the recombinant plasmids pET-30a-RAP- a mixture of these sera was used as the positive con- 1a61-1 and pET-30a-RAP-1a61-1/CT, which contained the trol serum. full-length gene (1359 bp, except for signal peptide se- Sera were collected from 492 lambs that were pur- quence) and the C-terminal-truncated fragment chased from a Babesia-free region in Jingtai country (450 bp), respectively. Both of the cloned RAP-1a (Gansu province of China) from 2010 to 2015. After proteins were tagged with histidine. nested PCR tests were performed, none of the samples tested positive, and a mixture of these serum samples Expression and purification of the recombinant proteins was used as the negative control serum to evaluate the To express the recombinant proteins, both pET-30a-RAP- specificity of the ELISA. 1a61-1 and pET-30a-RAP-1a61-1/CT were transformed Niu et al. Parasites & Vectors (2016) 9:313 Page 4 of 14

Fig. 1 The geographical distribution of the B. motasi-like sp. that was identified in this study and the distribution of Haemaphysalis spp. in China. The positive rates exceed 50 % indicated with deep red, and the positive rates ranged from 20 to 40 % indicated with light red, less than 10 % infected rates indicated without color into BL21 E. coli (DE3 strain) cells. The expression of the protein in Freund’s incomplete adjuvant (FIA, Sigma). His-tagged fusion proteins rRAP-1a61-1 and rRAP-1a61- These injections were administered on days 15, 20 and 1/CT was induced in the presence of kanamycin (50 μg/ 28. The sera were collected from the immunized rabbits ml) by adding 1 mM IPTG and then culturing the cells at 15 days after the last injection, purified and stored at 15 °C for 16 h. The bacterial cultures were harvested and -20 °C until further use. lysed using ultrasonication in binding buffer (50 mM Tris, 6 M Gu-HCl; pH 8.0) containing phenylmethylsulfonyl Western blot analysis fluoride (PMSF) and then purified as inclusion bodies from Approximately 20 μgofpurifiedBabesia sp. BQ1 (Lintan) the E. coli cells. The target protein was then eluted using recombinant proteins (rRAP-1a61-1 and rRAP-1a61-1/ urea followed by a series of buffers containing increasing CT) were separated using SDS-PAGE in 18 % polyacryl- concentrations of imidazole (200 mM, 500 m mM and amide gels. The proteins were transferred to nitrocellulose 2500 m mM). After the proteins were renatured, they were (NC) membranes with a 0.45 μm pore size (BioRad) at suspended in 1× SDS-PAGE sample loading buffer 24 V and 50 W for 35 min. The membrane was then cut (62.5 mM Tris-HCl [pH 6.8], 2 % SDS, 5 % β- into 0.25 cm strips and incubated in a blocking solution mercaptoethanol, 10 % glycerol, and 0.02 % bromophenol [5 % skimmed milk powder in Tris-buffered saline blue) and boiled for 5 min. The samples were briefly centri- (pH 7.6) with 0.1 % Tween-20 (TBST)] for approximately fuged prior to being separated using SDS-PAGE. Recom- 3 h at 4 °C on a shaker. After the NC strips had been binant protein expression was then analyzed using Western washed three times, they were incubated for 1 h with each blot analysis. tested serum (diluted at 1:100 in TBST). After the strips were washed three times in TBST, they were incubated for Preparation of anti-rRAP-1/CT specific rabbit immune serum 2 h with monoclonal anti-goat/sheep IgG-alkaline phos- New Zealand Rabbits (2 kg each) were subcutaneously phatase conjugates (Sigma, A8062, dilution: 1:5000). The injected with 400 μg of purified rRAP-1a61-1/CT pro- strips were then washed three times with TBST and tein that had been emulsified in Freund’s complete adju- incubated in a 5-bromo-4-chloro-3-indolyl phosphate/ vant (FCA, Sigma). In the negative control, the sera nitro blue tetrazolium (BCIP/NBT) liquid substrate were collected from each rabbit prior to the first injec- system (B1911-100ML, Sigma) for 15 to 20 min. The tions. Booster injections contained the same amount of approximate molecular weights of the revealed proteins Niu et al. Parasites & Vectors (2016) 9:313 Page 5 of 14

were evaluated by comparing their migration to a standard Results molecular weight marker (SM0671, 10–170 kDa, PageRuler Cloning and expression of RAP-1a61-1 and C-terminal- Prestained Protein Ladder; SM1861, 1.7–40 kDa, Spectra truncated RAP-1a61-1 Multicolor Low Range Protein Ladder, Thermo Scientific). The genes encoding RAP-1a61-1 (aa 22-474) and C- terminal RAP-1a61-1/CT (aa 325-474) (aa sequence IFAT and confocal laser microscopic observation corresponding to GenBank accession number AGV15809) An immunofluorescence analysis was performed using a were successfully amplified using PCR and inserted into procedure previously described by Moitra et al. [39] with the cloning vector pUC57. The pET-30a-RAP-1a61-1 and rabbit antiserum directed against rRAP-1a61-1/CT. The pET-30a-RAP-1a61-1/CT recombinant plasmids were iRBC membrane was stained using a PKH26 red fluores- identified using enzyme restriction analysis (Sac I/Sal I cent cell membrane labeling kit (MINI26, Sigma-Aldrich) and Mlu I/Hind III, respectively) and confirmed by according to the manufacturer’sinstructions.TheiRBCs sequencing using specific primers. The insert sequences were fixed in a buffer containing 4 % paraformaldehyde were also verified (data not shown). After the expression and 10 mM piperazine-N, N-bis (2-ethanesulfonic acid) in of each recombinant protein was induced using IPTG, PBS (PIPES) at pH 6.4 on 22 mm2 poly-L-lysine-coated the E. coli lysates were analyzed using SDS-PAGE and coverslips for 30 min at room temperature. The coverslips Western blot analysis of an anti-His serum. The results of were washed in PBS and then blocked for 1 h in 3 % SDS-PAGE showed that rRAP-1a61-1 and rRAP-1a61-1/ bovine serum albumin (BSA) in PBS containing 0.25 % Tri- CT were expressed as proteins that were approximately ton X-100. The coverslips were stained overnight at 4 °C 52 kDa and 20 kDa in size, respectively (Fig. 2). with either pre-immune serum or purified anti-rRAP- 1a61-1/CT serum (1:50) diluted in blocking buffer without Evaluation of the specificity of rRAP-1a61-1 and Triton X-100. The coverslips were washed 3 times in PBS, rRAP-1a61-1/CT using Western blot analysis stained for 1 h with FITC-conjugated anti-rabbit IgG The reactivity of each positive serum that was obtained (whole molecule) secondary antibodies (Sigma: F0382; from infected sheep with rRAP-1a61-1 and rRAP-1a61- 1:80), and then washed 3 times in PBS. The coverslips were 1/CT was analyzed using Western blot analysis. Strong stained with 1 μg/ml 4,6-diamidino-2-phenylindole’ (DAPI) reactions showing an expected size of rRAP-1a61-1/CT (Sigma: D8417) for 10 min and then examined for reactiv- (approximately 20 kDa) were observed in the positive ity using confocal microscopy. sera obtained from sheep infected with Babesia sp. BQ1 (Lintan) (No. 3216), Babesia sp. BQ1 (Ningxian) (No. ELISA 08026) and Babesia sp. Tianzhu (No. 08040). In con- The optimum concentration of coating buffer (0.1 M car- trast, no reaction was observed between rRAP-1a61-1/ bonate/bicarbonate, pH 9.6) was determined for rRAP- CT and sera obtained from sheep infected with Babesia 1a61-1/CT (100 μlat2μg/ml). It was then distributed and sp. Hebei (No. 08020) and Babesia sp. Xinjiang (No. adsorbed in 96-well flat-bottom microplates at 4 °C over- 3201) (Fig. 3). Furthermore, cross-reactivity between the night. After three washes with PBST (0.1 % Tween-20), the full-length rRAP-1a61-1 protein and sera from Babesia plates were blocked with 200 μl of 2 % gelatin in PBST at sp. Xinjiang-infected sheep and B. bigemina-infected cat- 37 °C for 1 h. After the plates were washed, the positive tle was also observed (data not shown). and negative sera (dilution: 1/100) were distributed in du- plicate, and the plates were incubated for 1 h at 37 °C. After Identification of native RAP-1 proteins using Western blot the plates were washed 3 times, 100 μl of monoclonal anti- analysis and IFAT goat/sheep IgG-peroxidase (Sigma, A9452, dilution: 1:1000) To identify native RAP-1 in Babesia sp. BQ1 (Lintan) was added to each well, and the wells were incubated for merozoites, antibodies against the C-terminal-truncated 1 h at 37 °C. After the plates were washed another 3 times, recombinant RAP-1a61-1/CT were obtained from rab- 100 μlof1-Step™ Ultra TMB-ELISA (34028–250 ml, bits and the positive serum obtained from animals in- Thermo Scientific) was added to each well, and the plates fected with Babesia sp. BQ1 (Lintan) (No. 3216), and were incubated for 15 to 25 min at room temperature. The the RAP-1a61-1 protein was detected at approximately reaction was stopped by adding 100 μlof2MH2SO4,and 52 kDa in the lysates of Babesia sp. BQ1 (Lintan)-in- the plates were read at 450 nm using an ELISA automat fected sheep erythrocytes (Fig. 4). No specific reaction (Bio-Rad Model 680 microplate reader, USA). was observed in Western blot analyses between the sera with the lysates of uninfected sheep erythrocytes (data Statistical analysis not shown). The 95 % confidence intervals (95 % CIs) for the overall The IFAT results showed that the merozoite nucleus of prevalence values of Babesia sp. BQ1 (Lintan) infection parasites were stained with DAPI and FITC-conjugated were calculated using IBM SPSS Statistics version 19.0. anti-rabbit IgG is able to detect anti-rRAP-1a61-1/CT Niu et al. Parasites & Vectors (2016) 9:313 Page 6 of 14

Cross-reactivity with other hemoparasites using ELISA Positive sera against Babesia sp. BQ1 (Lintan), Babesia sp. BQ1 (Ningxian), Babesia sp. Tianzhu, Babesia sp. Hebei, Babesia sp. Xinjiang, T. lunwenshuni, T. uilenbergi, A. ovis, B. bovis, and B. bigemina were tested using ELISA to evaluate cross-reactivity with rRAP-1a61-1/ CT. The tests were repeated in triplicate, and the specific antibody mean rate (AbR % = (Sample mean OD-Negative control mean OD)/(Positive control mean OD-Negative control mean OD) × 100) and standard deviation were calculated using Excel 2007 [38]. The results indicated that cross-reactivity was observed only between three B. motasi-like isolates (Lintan, Tianzhu and Ningxian) and that no cross-reactivity was observed with the sera against Babesia sp. Hebei, T. lunwenshuni, T. uilenbergi, B. bovis, B. bigemina or A. ovis (Fig. 6).

The kinetics of anti-Babesia sp. BQ1 (Lintan) antibodies were tested in experimentally infected sheep using ELISA The kinetics of antibodies produced against RAP-1a have been studied using ELISA with rRAP-1a61-1/CT in sera collected from 5 intact infected sheep (Nos. 3216, 2007, 3390, 3533 and 3446) before (0 weeks post- infection, WPI0) and after infection (from WPI1 to WPI12). A significant increase in the amount of antibodies produced against RAP-1a was observed after the sheep were infected, but varying kinetics were observed between individual sheep. In two of the Chinese sheep (Nos. 3216 and 2007), antibody production against RAP- Fig. 2 SDS-PAGE and Western blot analysis of the purified rRAP- 1a was increased during the early phase of infection, at 1a61-1 (Panel a and b) and C-terminal truncated rRAP-1a61-1/CT – (Panel c and d) proteins with His-tags that were expressed in E. coli 1 3 weeks (WPI1-3), after which it decreased. In con- cultures. M: molecular weight marker. Lanes 1 and 3: SDS-PAGE analysis; trast, a gradual increase in the number of antibodies Lanes 2 and 4: Western blot analysis using an anti-His antibody produced against RAP-1a was observed in the three French sheep (Nos. 3390, 3533 and 3446). Higher anti- antiserum. The immunofluorescence signals are the result body levels were observed at 6–7 weeks post-infection of the interaction between the serum from a rabbit that (WPI6-7) in sheep Nos. 3390 and 3446 and at 9 weeks was immunized by rRAP-1a61-1/CT and Babesia sp. BQ1 (WPI9) in sheep No. 3533. In general, antibodies were (Lintan) merozoites. Specific fluorescent signaling was not produced at 1 week post-inoculation, continued to in- detected using pre-immune rabbit serum (Fig. 5). crease through 6 weeks (WPI6), and remained stable

Fig. 3 Western blot analysis of the recombinant RAP-1a61-1/CT protein in Babesia sp. BQ1 (Lintan). M: molecular weight marker. Lanes 1 and 2: positive sera for Babesia sp. BQ1 (Lintan) (WPI3 and 4); Lanes 3 and 4: positive sera for Babesia sp. Tianzhu (WPI3 and 4); Lanes 5 and 6: positive sera for Babesia sp. BQ1 (Ningxian) (WPI3 and 4); Lanes 7 and 8: positive sera for Babesia sp. Hebei (WPI3 and 4); Lanes 9 and 10: positive sera for Babesia sp. Xinjiang (WPI3 and 4); Lanes 11-13: positive sera for B. bigemina, B. bovis and T. luwenshuni; and Lane 14: pre-immunized sheep (No. 3216) sera were used as the negative controls Niu et al. Parasites & Vectors (2016) 9:313 Page 7 of 14

until 9 weeks (WPI9), after which slight decreases were observed through 12 weeks (WPI9-12). High levels of antibodies against RAP-1a usually lasted 3 months (84 days p.i.), despite the decrease in production that was observed after 6 weeks (42 days p.i.) (Fig. 7).

Evaluation of specificity, sensitivity and positive threshold values in ELISA to detect rRAP-1/CT MedCalc statistical software was used to evaluate the sensitivity, specificity, and positive threshold value in ELISAs performed to test 191 positive sera and 492 negative sera, as previously described [40]. A percentage corresponding to the specific antibody mean rate (AbR %) was calculated for each serum sample. The results indicated that the threshold value was 18.84 %, which corresponded to 94.8 % sensitivity (95 % CI = 82.4– 98.4) and 96.1 % specificity (95 % CI = 85.1–99.4). The numbers of false positive and false negative sera were 10 and 15, respectively (Fig. 8).

ELISA identification of the rRAP-1a61-1/CT protein as a Fig. 4 Western blot analysis of native RAP-1 in Babesia sp. BQ1 potential antigen for use in serological epidemiology in (Lintan) merozoite lysates. Lysates from Babesia sp. BQ1 (Lintan)- B. motasi-like sub-group (Lintan, Ningxian and Tianzhu infected sheep erythrocytes were recognized by the serum of a isolates) infections rabbit that was immunized using rRAP-1a61-1/CT. Positive serum from Babesia sp. BQ1 (Lintan) (No. 3216) (Lanes 1 and 2). Pre-immunized Field serum samples were extracted from a total of 3198 sheep (No. 3216) and rabbit sera (Lanes 3 and 4) were used as the sheep and goats from 54 different regions of 23 Chinese negative controls provinces. The samples were analyzed using ELISA to evaluate the prevalence of infection with B. motasi-like

Fig. 5 Identification of native RAP-1 proteins on Babesia sp. BQ1 (Lintan) parasites using immunofluorescence analysis. Babesia sp. BQ1 (Lintan)- infected red blood cells were stained with rabbit pre-immune serum (control) and anti-RAP-1a61-1/CT antibodies. RBCs were stained with PKH26 (shown in red). An anti-RAP-1a61-1 antibody (green) reacted with native RAP-1a on merozoites. Nuclei were counterstained with DAPI (blue). Merged images are shown to the right. Arrowheads indicate the merozoites. Scale-bars: 7.5 μm Niu et al. Parasites & Vectors (2016) 9:313 Page 8 of 14

Fig. 6 The cross-reactivity of rRAP-1a61-1/CT from Babesia sp. BQ1 (Lintan) with positive sera against Babesia-, Theileria- and Anaplasma-infected sheep or cattle was determined using ELISA. LT: Babesia sp. BQ1 (Lintan), TZ: Babesia sp. Tianzhu, NX: Babesia sp. BQ1 (Ningxian), HB: Babesia sp. Hebei, XJ: Babesia sp. Xinjiang, T.l: T. luwenshuni, T.u: T. uilenbergi, A.o: A. ovis, B.bi: B. bigemina and B. bo: B. bovis

(Lintan, Ningxian and Tianzhu) isolates in these regions. in Tibet (2.22 %), Jilin (7.55 %) and Guangxi provinces The results of ELISA detection to identify positive sam- (9.74 %). Positive rates that were greater than 80 % were ples are summarized in Table 1. Sero-positive with B. observed in Chifeng (86.15 %) in Inner Mongolia and motasi-like were identified in 51 regions covering all 23 Lintan (83.33 %) in the Gansu provinces (Table 1, Fig. 1). surveyed provinces. The total positive rate in these regions was 36.02 % (95 % CI = 33.7–68.4). The sero- Discussion prevalence ranged from 2.22 to 81.11 %. The signifi- In the genus Babesia, RAP-1 was initially described cantly high positive rates exceeded 50 % in 7 of the 23 according to its recognition by monoclonal antibodies to surveyed provinces, including Liaoning (50 %), Inner merozoite antigens in B. bigemina. It was designated p58 Mongolia (61.01 %), Shaanxi (75.68 %), Hunan (52.73 %), at that time [41], and it was subsequently identified in all Sichuan (59.72 %), Anhui (81.11 %) and Shanxi (58 %). Babesia species that were examined. The sequence of the Three additional provinces, including Qinghai (45.35 %), full-length rap-1 gene was first published in B. bovis Ningxia (49.38 %) and Guangdong (41.89 %), had positive (named pBv60) [18], and the sequence and multigene rates exceeding 40 %. Lower sero-prevalence was observed nature of p58 (later named rap-1a)weredescribedinB.

Fig. 7 Kinetics of anti-Babesia sp. BQ1 (Lintan) antibodies in 5 sheep (Nos. 3216, 2007, 3446, 3390 and 3533) that were experimentally infected. The error bars indicate the standard deviations, and the bold dotted line shows the mean AbR that corresponded to each time point Niu et al. Parasites & Vectors (2016) 9:313 Page 9 of 14

were predicted in the N-terminal region (data not shown). Interestingly, the amino acid sequences between RAP-1a61 and RAP-1a67 are more conserved at the C-terminal region than the N-terminal region in B. motasi-like [34], which is not consistent with the common features described above. Moreover, the sequences of RAP-1a and the full rap-1 locus, including the intergenic regions in three isolates (Lintan, Ningxian and Tianzhu) of B. motasi-like, were almost identical [12]. The features of the more conserved C- terminal region of the RAP-1a protein in B. motasi- like could indicate that this form is an ideal candidate for diagnostics aimed at identifying several B. motasi- Fig. 8 Evaluation of the specificity, sensitivity and positive threshold like isolates. value of rRAP-1/CT in ELISA using antibody mean rates (AbRs) for The sera were able to recognize the native RAP-1 pro- positive and negative sera. 0 = negative sera, and 1 = positive sera tein in Western blot analysis and IFAT, suggesting that the sheep had been immunized against RAP-1 during bigemina [42]. The name RAP-1 was first applied when B. infection. Significant cross-reactivity was observed be- bovis pBv60 was found to be localized in rhoptries under tween rRAP-1a61-1/CT of Babesia sp. BQ1 (Lintan) and immunoelectron microscopy [27]. The definitive structure the positive sera obtained from Babesia sp. Tianzhu and of the rap-1 locus was initially defined in B. bovis [43] and Babesia sp. BQ1 (Ningxian) in both Western blot ana- then in B. bigemina [44]. In summary, an antibody react- lyses and ELISA, but not in the Hebei isolate. This is not ing with a B. bigemina protein (p58) that was later identi- surprising because the rap-1a sequences were almost fied as rap-1 was reported before its B. bovis homologue, identical in these three Babesia isolates at both the nu- and the sequence features, the association of these pro- cleotide and amino acid level. In contrast, the sequences teins with rhoptries, and the detailed organization of the of all of the rap-1 genes obtained from Babesia sp. locus containing rap-1 were initially described in B. bovis. Hebei were clearly different from the other 3 isolates, In all studied Babesia species, the RAP-1 proteins share suggesting that this isolate might belong to a different common conserved features that include the strict conser- species [12]. These findings were inconsistent with a vation of 4 cysteine residues, a highly conserved 14 amino previous study that indicated that weak cross-reactivity acid motif and several shorter conserved oligopeptide mo- between Babesia sp. BQ1 (Ningxian) and Babesia sp. tifs in the first N-terminal 300 amino acids. Among babe- BQ1 (Lintan) was detected using Western blot analysis sial parasites, the N-terminal regions of RAP-1 are more but not ELISA using a merozoite antigen (BQMA) [40]. conserved than those in the C-terminal region [32, 35]. Some novel antigens (e.g., BQP35 and BQHsp90) for In bovine Babesia species, previous studies have indi- Babesia sp. BQ1 (Lintan) were recently identified as can- cated that the full-length recombinant RAP-1a in B. didate diagnostic antigens. Western blot analysis and bovis could be used as a diagnostic antigen in ELISA to ELISA tests that are based on BQP35 have shown that detect antibodies and to show cross- reactivity with there is cross-reactivity between Babesia sp. BQ1 (Lintan) B. bigemina because of the conserved features in the and Babesia sp. Tianzhu, which is in agreement with the N-terminus of RAP-1, as described above [29]. In results obtained when merozoite extracts were used [51]. contrast, RAP-1 B-cell epitopes in B. bovis and B. However, rBQHSP90 could not differentiate between any bigemina have been mapped using monoclonal anti- B. motasi-like isolates and Babesia sp. Xinjiang, another bodies and immune sera obtained from cattle, and distinct ovine Babesia species [52]. Differences in cross- the results demonstrated that the B-cell epitopes pri- reactivity have been observed when using the ELISA marily targeted the C-terminal region of the proteins method based on BQP35, BQHsp90 or RAP-1a of Babesia [27, 45, 46]. Moreover, an ELISA based on recombin- sp. BQ1 (Lintan), and these might be caused by differences antRAP-1athatismodifiedintheC-terminalregion in molecular characterizations and the detection limita- has been developed to improve its sensitivity and spe- tions and sensitivities of these antigens. These findings cificity for antibody detection and could be widely suggest that rRAP-1a61-1/CT could be used as a poten- used as an antigen for serodiagnosis and epidemio- tially diagnostic antigen to increase the sensitivity and spe- logical survey purposes [37, 47–50]. In B. motasi-like cificity of such tests, and that it might replace native crude isolates, software prediction of RAP-1a B-cell epitopes parasite antigens, which require parasite reproduction demonstrated that B-cell epitopes were also located in (from the killing of experimentally infected animals) or the C-terminal region. Only a few B cell-epitopes the in vitro harvesting of cultures [53, 54]. Niu et al. Parasites & Vectors (2016) 9:313 Page 10 of 14

Table 1 Prevalence of Babesia sp. BQ1 Lintan in field samples collected from 23 provinces by detected antibodies produced from rRAP-1a CT with ELISA Province Prefecture No. of sera No. of positive sera Positive rate (%) in different Positive rate (%) in each regions province Qinghai Haibei 269 122 (122/269) 45.35 Xinjiang Yili 229 82 35.81 (118/419) 28.16 Akesu 90 20 22.22 Habahe 100 16 16 Gansu Jingtai 120 8 6.67 (160/447) 35.79 Jiayuguan 81 6 7.41 Tianzhu 81 56 69.14 Yongchang 75 15 20 Lintan 90 75 83.33 Tibet Lhasa 90 2 (2/90) 2.22 Liaoning Huanren 29 14 48.28 (42/84) 50 Anshan 27 10 37.04 Fengcheng 28 18 64.29 Jilin Yongji 26 3 11.54 (4/53) 7.55 Jiutai 27 1 3.70 Inner Mongolia Ordos 63 21 33.33 (133/218) 61.01 Manzhouli 13 0 0 Baotou 12 0 0 Chifeng 130 112 86.15 Shaanxi Yulin 74 56 (56/74) 75.68 Ningxia Wuzhong 81 40 (40/81) 49.38 Guangxi Jingxi 35 3 8.57 (15/154) 9.74 Tianyang 56 10 17.85 Pingxiang 18 0 0 Guilin 45 2 4.44 Hunan Xinhuang 29 16 55.17 (29/55) 52.73 Xiangtan 26 13 50 Sichuan Panzhihua 36 25 69.44 (43/72) 59.72 Hejiang 36 18 50 Yunnan Fuyuan 35 2 5.71 (65/202) 32.18 Honghe 32 16 50 Jinghong 20 5 25 Yanshan 34 10 29.41 Menghai 12 3 25 Ruili 32 14 43.75 Kunming 37 15 40.54 Anhui Hefei 90 73 (73/90) 81.11 Guangdong Zhaoqing 38 20 52.63 (31/74) 41.89 Lianzhou 36 11 30.56 Guizhou Dushan 30 1 3.33 (46/166) 27.71 Ziyun 32 13 40.63 Yuping 32 19 59.38 Guiyang 72 13 18.06 Niu et al. Parasites & Vectors (2016) 9:313 Page 11 of 14

Table 1 Prevalence of Babesia sp. BQ1 Lintan in field samples collected from 23 provinces by detected antibodies produced from rRAP-1a CT with ELISA (Continued) Province Prefecture No. of sera No. of positive sera Positive rate (%) in different Positive rate (%) in each regions province Chongqing Jiangjin 30 11 36.67 (15/53) 28.30 Wanzhou 23 4 17.39 Shandong Dongying 90 19 (19/90) 21.11 Shanxi Lvliang 50 29 (29/50) 58 Zhejiang Jingning 36 15 41.67 (32/117) 27.35 Qingtian 29 4 13.79 Hangzhou 52 13 25 Hubei Suizhou 81 27 (27/81) 33.33 Hebei Baoding 169 25 (25/169) 14.79 Henan Linzhou 60 20 33.33 (26/90) 28.89 Neihuang 30 6 20 Total 3198 1152 (1152/3198) 36.02

The kinetics analysis revealed that the humoral im- erythrocytes in a number of Babesia species [55]. How- mune response to this parasite varied in different sheep ever, studies focusing on the development of a serological breeds (Chinese and French sheep) during the course of diagnostic ELISA test that is based on recombinant infection. This result was inconsistent with a previous immune antigens that can detect ovine babesiosis are rare. study that used BQMA and indicated that there is no The development of ELISA tests requires preliminary difference in kinetics between these two sheep breeds knowledge of parasite polymorphism, the ability to suc- [38]. Antibody production against RAP-1 was initially in- cessfully culture cells in vitro to produce antigens, and/or creased in the two infected Chinese sheep between prior genetic knowledge of the targeted antigen so that WPI1 and WPI2, but it then decreased. These results specific sequences can be selected as an epitope. Some were consistent with the findings of a recent study, in improved ELISA methods for sero-epidemiological sur- which a RAP-1a peptide designed from the RAP-1a N- veys have been developed based on specific recombinant terminal of Babesia sp. BQ1 (Lintan) was used as an proteins (RAP-1 and SBPs) that are involved in the RBC antigen to perform ELISA. Antibody production against invasion process in B. bovis, B. bigemina and B. caballi this peptide displayed kinetics that were similar to those species [36, 37, 50, 56]. In this study, the results of sero- observed against rRAP-1a61-1 (data not shown). In con- investigations using an ELISA method that was developed trast, the production of specific antibodies against Babe- based on the rRAP-1a61-1/CT protein indicated that sia sp. BQ1 (Lintan) in the three infected French sheep Babesia sp. BQ1 (Lintan), Babesia sp. BQ1 (Ningxian) was increased at three weeks post-infection and peaked and Babesia sp. Tianzhu infections were identified in between six and nine weeks post-infection. On average, all of the investigated provinces. Recent large-scale sero- RAP-1a was expressed at sequentially higher levels from epidemiological ELISA surveys of Babesia sp. BQ1 week 1 to week 7 post-infection before its levels subse- (Lintan) were performed in small ruminants from 22 quently decreased. The difference in the kinetics of anti- provinces of China, and these were also regions that body production between these five infected sheep might were tested in our study, and the results indicated result from differences in sheep breeds or individual vari- that infection by this isolate was identified in all 22 ation. In general, our results are similar to those described investigated provinces, with an average prevalence of in a previous study that used a merozoite antigen (BQMA) 43.5 %. These values are slightly higher than the to evaluate the kinetics of antibody production during the values produced in our study and they showed a posi- course of Babesia sp. BQ1 (Lintan) infection. That study tive rate that was as high as 91 % in Inner Mongolia showed that antibody production was decreased from [57].Thepositiveratesfrom8provincesthatwere WPI 12 (approximately 80 DPI) [38]. tested using rRAP-1a were slightly higher, whereas A serological diagnostic test (ELISA) is the most prac- thepositiveratesfrom14oftheprovincesweresig- tical and economical assay for a wide range of epidemio- nificantly lower in our study than in a previous study logical investigative purposes, and it has been established [57]. This variation may result from differences in the as a method for evaluating the lysates of Babesia-infected limitations of the tested antigens. Moreover, another Niu et al. Parasites & Vectors (2016) 9:313 Page 12 of 14

sero-epidemiological investigation performed using study indicate that this ELISA is a potential diagnostic ELISA to detect infections in small ruminants by method for epidemiological investigations of known Babesia species indicated that there was an average related isolates in addition to other unidentified isolates in infection rate of 82.38 % for Babesia sp. BQ1 (Lintan) sheep B. motasi-like infections. in 261 sera samples obtained from Lintan County in Gansu province, and these result were similar to our Conclusions results (83.33 %) for this region [38]. The present study demonstrates that antibodies against The positive rates from 10 of the 23 provinces were rRAP-1a61-1 can detect native RAP-1 in Babesia sp. comparable to those found in a recent molecular detec- BQ1 (Lintan) merozoite lysates in Western blot analysis tion study that used a nested PCR assay based on the rap- and IFAT. The high conservation of RAP-1a across the 1b gene of B. motasi-like species, the positive rates for three isolates from B. motasi-like parasites suggests that Babesia sp. BQ1 (Lintan), Babesia sp. BQ1 (Ningxian) and rRAP-1a61-1/CT could be a promising common diag- Babesia sp. Tianzhu were 5, 13.8 and 21.9 % in Inner nostic antigen that can detect geographically distinct Mongolia, Gansu and Xinjiang, respectively in this previ- isolates of B. motasi-like infections in sheep. ous study (they were 61.01, 35.79 and 28.16 %, respectively, in our study) [58]. No B. motasi-like infections were Acknowledgements detected in DNA samples obtained from the Zhejiang, This study was financially supported by the NSFC (Nos. 31502054, 31372432, 31201899, 31272556, 31402189, and 31471967), ASTIP, Creative Research Guizhou, Chongqing and Liaoning provinces when the Groups of Gansu Province (No. 1210RJIA006), NBCIS CARS-38, the Special nested PCR test was used [58]. Larger variation was Fund for Agro-scientific Research in Public Research (Nos. 201303035 and observed in the numbers of positive samples that were 201303037), MOA, the 973 Program (2015CB150300), Supporting Program (2013BAD12B03, 2013BAD12B05), MOST (China), and the Jiangsu identified in these provinces when ELISA was used in our Co-innovation Center Programme for the Prevention and Control of study. A similar study was performed that compared the Important Animal Infectious Diseases and Zoonoses as a State Key results of using ELISA (66.84 %) and the molecular LAMP Laboratory of Veterinary Etiological Biology Project. method (14.3 %) to analyze Babesia sp. BQ1 (Lintan) Authors’ contributions infections in Gansu province [38]. Therefore, the differ- QN, JY, PY, YP and BZ performed the experiments, including cloning, ence in prevalence that is observed when using different expression, Western blot analysis, ELISA and IFAT. QN drafted the manuscript. ZL, EM, JL, GG and HY supervised all work. All authors read and approved testing methods (e.g., PCR vs ELISA or LAMP vs ELISA) the final version of the manuscript. or different antigens (e.g., BQMA vs rRAP-1a) or when using the same method (e.g., ELISA) can be potentially Competing interests explained by the parameters that were used or the limita- The authors declare that they have no competing interests. tions of the detection methods used in these assays. Author details Some studies have indicated that Haemaphysalis 1State Key Laboratory of Veterinary Etiological Biology, Key Laboratory of qinghaiensis and H. longicornis are the vectors of the Veterinary Parasitology of Gansu Province, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Science, Xujiaping 1, Lanzhou, Babesia sp. BQ1 (Lintan) isolate, while H. longicornis Gansu 730046, P. R. China. 2Jiangsu Co-innovation Center for Prevention and has been proposed as the transmission vector for Babe- Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou sia sp. BQ1 (Ningxian). These two tick species are 225009, P. R. China. 3INRA, UMR1300 Biology, Epidemiology and Risk Analysis in Animal Health, BP 40706F-44307 Nantes, France. widely distributed in at least 20 provinces of China (Fig. 1) [4, 59, 60]. Whereas, the higher positive rates Received: 2 March 2016 Accepted: 5 May 2016 (exceed 25 %) of B. motasi-like were found in Inner Mongolia, Xinjiang, Hunan, Zhejiang, Guangdong and References Chongqing, as well as in Guangxi with positive rate of 1. Friedhoff KT. Tick-borne diseases of sheep and goats caused by Babesia, 9.74 % where the H. longicornis and H. qinghaiensis have Theileria or Anaplasma spp. Parassitologia. 1997;39:99–109. not been reported yet. This has been explained by sev- 2. Guan GQ, Yin H, Luo JX, Lu WS, Zhang QC, Ma ML, et al. 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Liu AH, Yin H, Guan GQ, Schnittger L, Liu ZJ, Ma ML, et al. At least two between these three isolates is not surprising, and these genetically distinct large Babesia species infective to sheep and goats in isolates co-circulate in China with high prevalence. The China. Vet Parasitol. 2007;147:246–51. 7. Niu QL, Luo JX, Guan GQ, Liu ZJ, Ma ML, Liu AH, et al. Differentiation of two results suggest that the development of ELISA methods ovine Babesia based on the ribosomal DNA internal transcribed spacer (ITS) that are based on the rRAP-1a61-1/CT described in our sequences. Exp Parasitol. 2009;121:64–8. Niu et al. Parasites & Vectors (2016) 9:313 Page 13 of 14

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